Abstract
OBJECTIVE: To evaluate the feasibility of using cell membrane vesicles (CMVs) as a delivery system for small interfering RNA (siRNA) and to assess their performance in a lipopolysaccharide (LPS)-induced inflammatory model of human dental pulp stem cells (DPSCs). METHODS: CMVs were generated from cytochalasin B-treated 3T3 cells and characterized for their physicochemical properties, including morphology, size distribution, and zeta potential, using confocal microscopy and dynamic light scattering. To construct CMVs@siTNF-α, saponin-mediated transient permeabilization was used to facilitate siRNA loading, after which encapsulation efficiency was evaluated by flow cytometry and confocal imaging. Intracellular uptake behaviors were examined using flow cytometry in DPSCs. LPS (1 mg/L) was employed to establish a robust in vitro inflammatory microenvironment. DPSCs were subsequently treated with CMVs or CMVs@siTNF-α, and cell viability was assessed via CCK-8 (cell counting kit-8). The expression and secretion of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme linked immunosorbent assay (ELISA) to evaluate both gene-silencing efficiency and downstream anti-inflammatory effects. RESULTS: CMVs exhibited uniform spherical morphology with an average diameter of approximately 903 nm and a zeta potential of -9.39 mV, confirming successful vesicle formation. CMVs efficiently encapsulated FAM-labeled siRNA, as indicated by a pronounced fluorescence shift in flow cytometry and clear colocalization signals in confocal imaging. DPSCs cultured with CMVs@FAM-siTNF-α demonstrated increased intracellular fluorescence, reflecting efficient vesicle uptake and cytoplasmic siRNA release. Importantly, both CMVs and CMVs@siTNF-α displayed negligible cytotoxicity. LPS stimulation significantly elevated TNF-α, IL-1β, and IL-6 expression, validating the inflammatory model. CMVs alone did not affect cytokine levels, indicating biological inertness. In contrast, CMVs@siTNF-α significantly suppressed the LPS-induced upregulation of TNF-α at both mRNA and protein levels., demonstrating potent gene-silencing activity. Furthermore, suppression of TNF-α led to downstream attenuation of IL-1β and IL-6, with both transcription and secretion significantly decreased compared with the LPS group. These findings collectively confirmed that CMVs enabled efficient intracellular siRNA transport and effectively mitigate inflammatory responses in DPSCs. CONCLUSION: CMVs represent a biocompatible and effective siRNA delivery platform capable of achieving robust TNF-α knockdown and ameliorating inflammatory cytokine production in vitro, highlighting their potential for future nucleic acid-based anti-inflammatory therapies.